The activation of porcine pancreatic lipase by cis-unsaturated fatty acids.

In the presence of taurodeoxycholate, cis-unsaturated fatty acids increase porcine pancreatic lipase activity 15-fold at pH 7.5. This effect is saturable with a low proportion of fatty acid to substrate. The overall angle of the fatty acid, the position of its double bond and the presence of a carboxyl group were critical factors in whether the fatty acid effectively increased lipase activity. When the substrate is emulsified by taurodeoxycholate, the pH optimum for lipase ranges from 6.2 to 7.0. In the presence of cis-unsaturated fatty acids, the overall activity of lipase increases, the pH optimum shifts, and the pH-activity curve becomes biphasic, with one optimum around pH 7.7, and the other around pH 8.8. Fluorescence studies indicate that fatty acids bind near aromatic residues in lipase, particularly tryptophan. Using the fluorescent fatty acid cis-parinaric acid, it was determined that multiple binding sites are present with Kd values of approx. 10(-6) M. Far-UV circular dichroism (CD) studies indicate that in addition to a high affinity fatty acid binding site with a Kd of approx. 10(-6) M, there is also a low affinity binding site with a Kd of approx. 10(-4) M. The far-UV CD data also show that cis-unsaturated fatty acids change the conformation of lipase. It is calculated that the percentage of alpha helix decreases, and the amount of beta sheet and beta turn structure increases. Because the three-dimensional crystal structure of lipase is known, a model is proposed to describe how cis-unsaturated fatty acids increase lipase activity.

Action of a thionin isolated from nuts of Pyrularia pubera on human erythrocytes.

Pyrularia thionin is a strongly basic peptide of 47 amino acids isolated from Pyrularia pubera. This peptide, a member of the thionin family, is hemolytic, cytotoxic and neurotoxic. The characteristics of the hemolytic activity on human erythrocytes are as follows: (1) the peptide does not itself have any phospholipase activity in a micellar assay system with egg yolk phosphatidylcholine, as evidenced by a lack of pH change or uptake of oxygen in the presence of lipoxidase; (2) erythrocyte membranes treated with thionin, however, show a low level of oxygen uptake in the presence of lipoxidase as a consequence of fatty acid release, and this activity is synergistic with that of bee venom phospholipase A2; (3) hemolysis caused by thionin is synergistic with added bee venom phospholipase A2; (4) kinetic analysis of the hemolytic assay reveals that the reaction follows Michaelis-Menten kinetics, being saturable with thionin with a Km of 1.6 microM; (5) binding studies with 125I-thionin show by Scatchard analysis a Kd value of 2.1 microM; (6) although iodinated thionin is inactive in the hemolysis assay, it acts as a competitive inhibitor to native thionin in the hemolytic assay; the inhibitor constant, Ki, for this reaction is 7.0 microM; and (7) Ca2+ above 1 mM inhibits the reaction. All the data are consistent with thionin binding to a receptor, most likely a protein, on the erythrocyte membrane, leading to the release of free fatty acids, most likely by activation of phospholipase A2. The release of fatty acids is itself not sufficient to explain the hemolytic reaction.